Plant activation of Cry8Bb1 by insertion of a plant protease-sensitive site

ABSTRACT

Compositions and methods for protecting a plant from an insect pest are provided. In particular, nucleic acid sequences encoding insect protoxins modified to comprise at least one proteolytic activation site that is sensitive to a plant protease or an insect gut protease are provided. Cleavage of the modified protoxin at the proteolytic activation site by a protease produces an active insect toxin. Methods of using the modified insect protoxin nucleic acid sequences and the polypeptides they encode to protect a plant from an insect pest are provided. Particular embodiments of the invention further provide modified insect protoxin compositions and formulations, expression cassettes, and transformed plants, plant cells, and seeds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/532,185 filed on Dec. 23, 2003, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the fields of plant molecular biologyand plant pest control. More specifically, the present invention relatesto modified insect protoxins and the nucleic acid sequences that encodethem. Methods of the invention utilize these modified insect protoxinsand nucleic acid sequences to control plant pests.

BACKGROUND OF THE INVENTION

Insect pests are a major factor in the loss of the world's agriculturalcrops. For example, corn rootworm feeding damage or boll weevil damagecan be economically devastating to agricultural producers. Insectpest-related crop loss from corn rootworm alone has reached one billiondollars a year.

Traditionally, the primary methods for impacting insect pestpopulations, such as corn rootworm populations, are crop rotation andthe application of broad-spectrum synthetic chemical pesticides.However, consumers and government regulators alike are becomingincreasingly concerned with the environmental hazards associated withthe production and use of synthetic chemical pesticides. Because of suchconcerns, regulators have banned or limited the use of some of the morehazardous pesticides. Thus, there is substantial interest in developingalternatives to traditional chemical pesticides that present a lowerrisk of pollution and environmental hazards and provide a greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a broad range of insect pestsincluding Lepidoptera, Diptera, Coleoptera, Hemiptera, and others.Bacillus thuringiensis and Bacillus papilliae are among the mostsuccessful biocontrol agents discovered to date. Insect pathogenicityhas been attributed to strains of: B. larvae, B. lentimorbus, B.papilliae, B. sphaericus, B. thuringiensis (Harwook, ed. (1989) Bacillus(Plenum Press), p. 306) and B. cereus (International Publication No. WO96/10083). Pesticidal activity appears to be concentrated in parasporalcrystalline protein inclusions, although pesticidal proteins have alsobeen isolated from the vegetative growth stage of Bacillus. Severalgenes encoding these pesticidal proteins have been isolated andcharacterized (see, for example, U.S. Pat. Nos. 5,366,892 and5,840,868).

Microbial pesticides, particularly those obtained from Bacillus strains,have played an important role in agriculture as alternatives to chemicalpest control. Pesticidal proteins isolated from strains of Bacillusthuringiensis, known as δ-endotoxins or Cry toxins, are initiallyproduced in an inactive protoxin form. These protoxins areproteolytically converted into an active toxin through the action ofproteases in the insect gut. See, Rukmini et al. (2000) Biochimie82:109-116; Oppert (1999) Arch. Insect Biochem. Phys. 42:1-12 andCarroll et al. (1997) J. Invertebrate Pathology 70:41-49. Proteolyticactivation of the toxin can include the removal of the N- and C-terminalpeptides from the protein, as well as internal cleavage of the protein.Other proteases can degrade pesticidal proteins. See Oppert, ibid.; seealso U.S. Pat. Nos. 6,057,491 and 6,339,491. Once activated, the Crytoxin binds with high affinity to receptors on epithelial cells in theinsect gut, thereby creating leakage channels in the cell membrane,lysis of the insect gut, and subsequent insect death through starvationand septicemia. See, e.g., Li et al. (1991) Nature 353:815-821.

Recently, agricultural scientists have developed crop plants withenhanced insect resistance by genetically engineering crop plants toproduce pesticidal proteins from Bacillus. For example, corn and cottonplants genetically engineered to produce Cry toxins (see, e.g., Aronson(2002) Cell Mol. Life Sci. 59(3):417-425; Schnepf et al. (1998)Microbiol. Mol. Biol. Rev. 62(3):775-806) are now widely used inAmerican agriculture and have provided the farmer with anenvironmentally friendly alternative to traditional insect-controlmethods. In addition, potatoes genetically engineered to containpesticidal Cry toxins have been sold to the American farmer. Thepresence of endogenous proteases in plants that can degrade andinactivate the insect toxins expressed in these transgenic plants,however, limits the usefulness of these pest-control techniques.

Researchers have determined that plants express a variety of proteases,including serine and cysteine proteases. See, e.g., Goodfellow et al.(1993) Plant Physiol. 101:415-419; Pechan et al. (1999) Plant Mol. Biol.40:111-119; Lid et al. (2002) Proc. Nat. Acad. Sci. USA 99:5460-5465.Previous research has also shown that insect gut proteases includecathepsins, such as cathepsin B- and L-like proteinases. See, Shiba etal. (2001) Arch. Biochem. Biophys. 390:28-34; Purcell et al. (1992)Insect Biochem. Mol. Biol. 22:41-47. For example, cathepsin L-likedigestive cysteine proteinases are found in the larval midgut of Westerncorn rootworm. See, Koiwa et al. (2000) FEBS Letters 471:67-70; Koiwa etal. (2000) Analytical Biochemistry 282:153-155.

While investigators have previously genetically engineered plants,particularly crop plants, to contain biologically active (i.e.,pesticidal) Cry toxins, these foreign proteins can be degraded andinactivated by proteases present in these transgenic plants. Moreover,researchers to date have not effectively utilized the protoxin forms ofpesticidal polypeptides in conjunction with endogenous plant or insectgut proteases to control plant pests. Thus, new strategies for modifyinginsect toxins and utilizing these modified insect toxins in pestmanagement strategies are desired.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for protecting a plant from an insect pest areprovided. Compositions are novel nucleic acid molecules comprisingnucleotide sequences encoding insect protoxins that comprise at leastone proteolytic activation site that has been engineered to comprise acleavage site that is sensitive to cleavage by a plant protease or issensitive to cleavage by an insect gut protease. The proteolyticactivation site is engineered within the activation region of the insectprotoxin such that proteolytic cleavage by the plant protease or insectgut protease releases the activated insect toxin within a plant cell orwithin the insect gut, respectively. The novel nucleic acid moleculescan be operably linked to any promoter of interest to drive expressionof these modified insect protoxins in plant cells. Expression cassettesand transgenic plant cells, plants, and seeds comprising these novelnucleic acid molecules are also provided. Modified insect protoxins andmethods for their use in controlling plant pests are further provided.

The nucleic acid compositions of the invention are useful in methodsdirected to protecting plants from insect pests and in methods forimpacting insect pests. The methods comprise introducing into a plant apolynucleotide construct comprising a nucleotide sequence that encodes amodified insect protoxin operably linked to a promoter that drivesexpression in a plant cell. Where the modified insect protoxin comprisesa proteolytic activation site that is engineered to comprise a cleavagesite that is sensitive to a plant protease, expression of thepolynucleotide construct produces the modified insect protoxin in theplant cell, wherein it is cleaved by a plant protease to generate theactive insect toxin. The presence of the insect toxin protects the plantfrom an insect pest. Where the modified insect protoxin comprises aproteolytic activation site that is engineered to comprise a cleavagesite that is sensitive to an insect gut protease, expression of thepolynucleotide construct produces the modified insect protoxin withinthe cells of the transgenic plant. When a susceptible insect pest feedson the transgenic plant and, thus, also ingests the modified protoxinthat has been expressed in the plant, the modified insect protoxin iscleaved by an insect gut protease to generate the active toxin in theinsect gut, thereby impacting the insect pest.

The present invention further provides nucleic acid molecules encodingnovel insect gut proteases and biologically active variants andfragments thereof. The novel proteases are useful in methods directed toidentification of preferred proteolytic cleavage sites for these insectgut proteases. Having identified these preferred proteolytic cleavagesites, insect protoxins of interest can be modified to comprise thepreferred proteolytic cleavage sites within at least one proteolyticactivation site to improve activation of the insect protoxin within aninsect gut. Where an insect protoxin of interest alternatively or alsocomprises one or more of these preferred cleavage sites in a region ofthe protoxin that is outside an activation region but within theactivated insect toxin, the preferred cleavage site can be replaced witha proteolytic protection site to protect the insect toxin fromproteolytic inactivation in the insect gut.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compositions and methods thatprovide for protection of a plant from insect pests, and which can beutilized to impact these insect pests. The compositions are novelnucleic acid molecules comprising nucleotide sequences encoding modifiedinsect protoxins that provide for efficient processing into activetoxins either within the cells of a plant host that is capable ofexpressing the modified insect protoxin or within the gut of the insectpest that feeds on a plant host that is capable of expressing themodified insect protoxin.

“Modified insect protoxin” is intended to mean an insect protoxin thatcomprises at least one proteolytic activation site that is not naturallyoccurring within the insect protoxin, and which has been engineered tocomprise a cleavage site that either is sensitive to cleavage by a plantprotease residing within the cells of a plant, or is sensitive tocleavage by an insect gut protease. “Sensitive to cleavage” is intendedto mean that the protease recognizes the cleavage site, and thus iscapable of cleaving the protoxin at that cleavage site. In bothinstances, the non-naturally occurring proteolytic activation site isengineered within an activation region of the insect protoxin.“Activation region” is intended to mean a region within the insectprotoxin wherein proteolytic cleavage at the engineered activation siteresults in the production of a biologically active insect toxin. Forpurposes of the present invention, this biologically active insect toxinis also referred to as the “active insect toxin,” the “activated insecttoxin,” or the “activated form” of an insect protoxin.

The compositions of the invention also include polynucleotide constructscomprising these nucleic acid molecules. These constructs include, butare not limited to, expression cassettes, wherein the nucleotidesequences encoding the modified insect protoxins are operably linked toa promoter that drives expression in a plant cell. The invention furtherprovides plant cells, plants, and seeds stably transformed with thesepolynucleotide constructs. The compositions of the invention are usefulin protecting a plant from insect pests, and can be utilized to impactinsect pests that interact with a plant during one or more phases of theinsect life cycle.

In one embodiment, the novel nucleic acid molecules of the inventioncomprise nucleotide sequences that encode a modified insect protoxinthat comprises at least one proteolytic activation site that has beenengineered to comprise a cleavage site that is sensitive to cleavage bya plant protease as noted herein below. Such nucleic acid molecules canbe utilized in the methods of the invention to protect a plant frominsect pests. In this manner, a polynucleotide construct comprising thistype of modified insect protoxin coding sequence, operably linked to apromoter that drives expression in a plant cell, can be introduced intoa plant. Expression of this polynucleotide construct within cells ofthis plant produces the modified insect protoxin in those cells. Theinactive modified insect protoxin is then cleaved by a plant protease atthe engineered proteolytic activation site to produce a biologicallyactive insect toxin that protects the plant from an insect pest thatfeeds on cells of the plant comprising the active insect toxin.

In another embodiment, the novel nucleic acid molecules of the inventioncomprise nucleotide sequences encoding a modified insect protoxin thatcomprises at least one proteolytic activation site that has beenengineered to comprise a cleavage site that is sensitive to cleavage bya protease that resides within an insect gut. In some embodiments, theproteolytic activation site is engineered to comprise a cleavage sitethat is the preferred cleavage site for a novel insect gut proteasedisclosed herein below. Such nucleic acid molecules can be utilized inthe methods of the invention to impact insect pests. “Impact an insectpest” or “impacting an insect pest” is intended to mean, for example,deterring the insect pest from feeding further on the plant, harming theinsect pest, or killing the insect pest as noted herein below. In thismanner, a polynucleotide construct comprising this type of modifiedinsect protoxin coding sequence, operably linked to a promoter thatdrives expression in a plant cell, can be introduced into a plant.Expression of this polynucleotide construct within cells of this plantproduces the modified protoxin in those plant cells. When an insect pestfeeds on cells of the plant that are expressing this modified insectprotoxin, the ingested modified insect protoxin is cleaved by the insectgut protease, thereby producing an active insect toxin in the insect gutand impacting the insect pest. Cleavage can result in removal of theN-terminal sequence, the C-terminal sequence or both sequences. Inaddition to N- and C-terminal processing, part of the activation processmay also involve cleavage between the alpha 3 and alpha 4 helices.

In other embodiments, the invention is drawn to the modified insectprotoxins encoded by the nucleic acid molecules of the present inventionand to methods for using these polypeptides. Compositions andformulations comprising a modified insect protoxin, or variant orfragment thereof, that comprises at least one, non-naturally occurringproteolytic activation site that has been engineered to comprise acleavage site that is sensitive to cleavage by an insect gut protease,are useful in methods directed to impacting insect pests. In thismanner, the invention further provides a method for impacting an insectpest of a plant comprising applying, for example, a composition orformulation comprising this type of modified insect protoxin to theenvironment of the insect pest. In one embodiment, the modified insectprotoxin is combined with a carrier for subsequent application to theenvironment of the insect pest. While the invention is not bound by anytheory of operation, in one embodiment, an insect pest ingests themodified insect protoxin. The modified protoxin is then cleaved by aninsect gut protease to produce a biologically active toxin in the insectpest gut, thereby impacting the insect pest.

One of skill in the art would recognize that the compositions andmethods of the invention can be used alone or in combination with othercompositions and methods for controlling insect pests that impactplants. For example, the present invention may be used in conjunctionwith other pesticidal proteins or traditional chemical pesticides.

While the invention does not depend on a particular biological mechanismfor protecting a plant from an insect pest, expression of the nucleotidesequences of the invention in a plant can result in the production ofactive insect toxins that increase the resistance of the plant to insectpests. The transgenic plants of the invention find use in agriculture inmethods for protecting plants from insect pests and for impacting insectpests. Certain embodiments of the invention provide transformed cropplants, such as, for example, maize plants, which find use in methodsfor impacting insect pests of the plant, such as, for example, western,northern, southern, and Mexican corn rootworms. Other embodiments of theinvention provide transformed potato plants, which find use in methodsfor impacting the Colorado potato beetle, transformed cotton plants,which find use in methods for impacting the cotton boll weevil, andtransformed turf grasses, which find use in methods for impacting thebluegrass billbug, Sphenophorous parvulus.

“Insect protoxin” or “protoxin” is intended to mean a biologicallyinactive polypeptide that is converted to an active insect toxin uponcleavage at a proteolytic activation site by a protease. In someembodiments, activation of the toxin proceeds by removal of a C-terminalpeptide, an N-terminal peptide, or peptides from both the N-terminal andC-terminal regions of the protoxin. “Insect toxin” refers to theactivated form of an insect protoxin, i.e., the cleaved polypeptide thatpossesses pesticidal activity. As used herein, the term “pesticidalactivity” refers to activity of a substance, such as, for example, aprotein, that can be measured by routine assays known in the art. Suchassays include, but are not limited to, pest mortality, pest weightloss, pest repellency, pest attraction, and other behavioral andphysical changes of a pest after feeding and exposure to the substancefor an appropriate length of time. General procedures include additionof the experimental compound or organism to the diet source in anenclosed container. Assays for assessing pesticidal activity are wellknown in the art. See, e.g., U.S. Pat. Nos. 6,570,005 and 6,339,144;herein incorporated by reference in their entirety.

The preferred developmental stage for testing for pesticidal activity islarvae or immature forms of an insect of interest. The insects may bereared in total darkness at from about 20° C. to about 30° C. and fromabout 30% to about 70% relative humidity. Bioassays may be performed asdescribed in Czapla and Lang (1990) J. Econ. Entomol. 83(6):2480-2485.Methods of rearing insect larvae and performing bioassays are well knownto one of ordinary skill in the art.

In some embodiments of the invention, the insect toxin is a Bacillusthuringiensis (Bt) toxin. “Bt” or “Bacillus thuringiensis” toxin isintended to mean the broader class of toxins found in various strains ofBacillus thuringiensis, which includes such toxins as, for example, thevegetative insecticidal proteins and the δ-endotoxins. The vegetativeinsecticidal proteins (for example, members of the VIP1, VIP2, or VIP3classes) are secreted insecticidal proteins that undergo proteolyticprocessing by midgut insect fluids. They have pesticidal activityagainst a broad spectrum of Lepidopteran insects. See, for example, U.S.Pat. No. 5,877,012, herein incorporated by reference in its entirety.The Bt δ-endotoxins are toxic to larvae of a number of insect pests,including members of the Lepidoptera, Diptera, and Coleoptra orders.These insect protoxins include, but are not limited to, the crytoxins,including, for example, Cry 1, Cry 3, Cry 5, Cry 8, and Cry 9. Ofparticular interest are the Cry 8 or Cry 8-like δ-endotoxins. “Cry8-like” is intended to mean that the nucleotide or amino acid sequenceshares a high degree of sequence identity or similarity to previouslydescribed sequences categorized as Cry8, which includes such toxins as,for example, Cry8Bb1 (see Genbank Accession No. CAD57542) and Cry8Bc1(see Genbank Accession No. CAD57543). See co-pending U.S. patentapplication Ser. No. 10/666,320, filed Jun. 25, 2003, hereinincorporated by reference. “Cry8-like insect protoxin” is intended tomean the biologically inactive polypeptide that is converted to theactivated Cry8-like insect toxin upon cleavage at a proteolyticactivation site by a protease. It is the activated Cry8-like insecttoxin that has pesticidal activity. As used herein, “Cry8-like insecttoxin” refers to a biologically active pesticidal polypeptide thatshares a high degree of sequence identity or similarity to Cry8 insecttoxin sequences.

The Bt toxins are a family of insecticidal proteins that are synthesizedas protoxins and crystallize as parasporal inclusions. When ingested byan insect pest, the microcrystal structure is dissolved by the alkalinepH of the insect midgut, and the protoxin is cleaved by insect gutproteases to generate the active toxin. The activated Bt toxin binds toreceptors in the gut epithelium of the insect, causing membrane lesionsand associated swelling and lysis of the insect gut. Insect deathresults from starvation and septicemia. See, e.g., Li et al. (1991)Nature 353:815-821.

The protoxin form of the Cry toxins contains a crystalline formingsegment. A comparison of the amino acid sequences of active Cry toxinsof different specificities further reveals five highly-conservedsequence blocks. Structurally, the Cry toxins comprise three distinctdomains, which are, from the N- to C-terminus: a cluster of sevenalpha-helices implicated in pore formation (referred to as “domain 1”),three anti-parallel beta sheets implicated in cell binding (referred toas “domain 2”), and a beta sandwich (referred to as “domain 3”). Thelocation and properties of these domains are known to those of skill inthe art. See, for example, Li et al. (1991) supra and Morse et al.(2001) Structure 9:409-417.

The modified insect protoxins of the invention can be derived from anysuitable native (i.e., naturally occurring) insect protoxin, such as thenative Bt δ-endotoxins described above, by engineering the proteolyticactivation site of interest within the native insect protoxin sequence.In this manner, a nucleotide sequence encoding the native insectprotoxin of interest can be altered, for example, by site-directedmutagenesis, to comprise the codons for the proteolytic activation siteof interest, i.e., a site sensitive to plant proteases or a sitesensitive to insect gut proteases. As noted above, the codons for theproteolytic activation site(s) of interest are engineered within theregion of the native coding sequence that corresponds to the activationregion of the native insect protoxin, so that proteolytic cleavage ofthe encoded modified insect protoxin by the protease of interest resultsin production of the active insect toxin.

Alternatively, the modified insect protoxins of the invention can bederived from fragments or variants of native insect protoxins, asdefined herein below, so long as the fragment or variant of the nativeinsect protoxin yields an activated (i.e., having pesticidal activity)insect toxin upon proteolytic cleavage by the protease of interest(i.e., plant protease or insect gut protease). In this manner, thecoding sequences for such fragments and variants of the native insectprotoxin protein serve as the starting material for engineering in thecodons for the proteolytic activation site(s) of interest. In essence, amodified insect protoxin designed in this manner represents a fragmentor variant of the native insect protoxin that has been engineered tocomprise the proteolytic activation site of interest within theactivation region of the respective polypeptide.

It is recognized that variations in a modified insect protoxin disclosedherein can be introduced at the level of the nucleic acid molecule thatencodes a modified form of a native insect protoxin in order to producea variant of the encoded modified insect protoxin. That is, havingdisclosed a nucleotide sequence encoding a native insect protoxin withat least one proteolytic activation site of interest engineered withinthe native insect protoxin sequence, one of skill in the art cansubsequently introduce variations into the disclosed nucleotide sequenceof the invention, so that the encoded modified insect protoxin is avariant of the modified native insect protoxin. Such variations includedeletions, substitutions, and additions of one or more residues, andinclude variations that result in truncated forms of the modified insectprotoxin. Any such variations can be introduced into the nucleotidesequence encoding the modified native insect protoxin so long as theencoded variant of the modified insect protoxin can be cleaved toproduce a biologically active insect toxin, i.e., an insect toxin thathas pesticidal activity as noted elsewhere herein. Such variants andfragments are well-known in the art. See, e.g., co-pending U.S. patentapplication Ser. No. 10/606,320, filed Jun. 25, 2003; U.S. Pat. No.5,877,012; herein incorporated by reference in their entirety.

“Protecting a plant from an insect pest” is intended to mean limiting oreliminating insect pest-related damage to a plant by, for example,inhibiting the ability of the insect pest to grow, feed, and/orreproduce or by killing the insect pest.

As used herein, “impacting an insect pest of a plant” includes, but isnot limited to, deterring the insect pest from feeding further on theplant, harming the insect pest by, for example, inhibiting the abilityof the insect to grow, feed, and/or reproduce, or killing the insectpest.

A “protease” is intended to mean an enzyme that cleaves polypeptides byhydrolyzing peptide bonds. A “plant protease” is intended to mean aprotease that is naturally found in any plant of the invention. Previousresearch has shown that plants express a variety of proteases, includingserine and cysteine proteases. See, e.g., Goodfellow et al. (1993) PlantPhysiol. 101:415-419; Pechan et al. (1999) Plant Mol. Biol. 40:111-119;Lid et al. (2002) Proc. Nat. Acad. Sci. USA 99:5460-5465. Any plantprotease may be used in the present invention. In some embodiments, theplant protease is a cysteine protease, for example, a cathepsin orcathepsin-like protease. In one embodiment, the cysteine protease is acathepsin B-like protease.

As used herein, “insect gut protease” refers to a protease that isnaturally found in the digestive tract of an insect pest. Researchershave established that a wide array of proteases are expressed in theinsect gut, including cysteine and serine proteases. See, e.g., Shiba etal. (2001) Arch. Biochem. Biophys. 390:28-34; see also, Purcell et al.(1992) Insect Biochem. Mol. Biol. 22:1-47; Koiwa et al. (2000) FEBSLetters 471:67-70; Koiwa et al. (2000) Anal. Biochem. 282:153-155. Anyinsect gut protease may be used in the present invention. In someembodiments, the insect gut protease is a cysteine protease, forexample, a cathepsin B-like or cathepsin L-like protease. In otherembodiments, the insect gut protease is a serine protease, for example,trypsin or chymotrypsin.

A “proteolytic site” is intended to mean an amino acid sequence thatconfers sensitivity to a class of proteases or a particular proteasesuch that a polypeptide comprising the amino acid sequence is cleaved atthat site by members of the class of proteases or by the particularprotease. As used herein, a “proteolytic activation site” is aproteolytic site that has been engineered into an activation region ofan insect protoxin. As used herein, an “activation region” is a regionof an insect protoxin such that proteolytic cleavage at the proteolyticactivation site within the activation region generates a biologicallyactive insect toxin. A proteolytic site is said to be “sensitive” to theprotease(s) that recognizes that site. It is recognized that theefficiency of proteolytic digestion will vary, and that a decrease inefficiency of proteolytic digestion can lead to an increase in stabilityor longevity of the polypeptide within a plant cell or within an insectgut. Thus, a proteolytic site may confer sensitivity to more than oneprotease or class of proteases, but the efficiency of digestion at thatsite by various proteases may vary.

Proteolytic sites include, for example, trypsin sites, chymotrypsinsites, papain sites, cathepsin sites, and cathepsin-like sites.Proteolytic sites for particular proteases often comprise “motifs,” orsequence patterns, that are known to confer sensitivity to a particularprotease. Thus, for example, cathepsin site motifs include FRR, acathepsin L protease cleavage site; RR, a trypsin and cathepsin Bcleavage site; LKM, a chymotrypsin site; and FF, a cathepsin D site. Aputative proteolytic site is a sequence that comprises a motif orcomprises a sequence similar to a motif but which has not been shown tobe subject to digestion by the corresponding protease. In oneembodiment, the modified insect protoxins of the invention have aproteolytic activation site that comprises the motif FRRGFRRG (SEQ IDNO:6).

In some embodiments of the invention, the proteolytic activation site isintroduced in the C-terminal portion of the protoxin, the N-terminalportion of the protoxin, or in both the N-terminal and C-terminalregions. Likewise, in some embodiments, cleavage of the protoxin willresult in the removal of an N-terminal peptide, a C-terminal peptide, orpeptides from both the N-terminal and C-terminal regions of the protein.In one particular embodiment, the proteolytic activation site isintroduced in the junction between the N-terminal crystalline formingsegment of the protoxin and the C-terminal portion of the protoxin thatcomprises the active insect toxin upon cleavage.

It is further recognized that insect toxins expressed in a plant cellmay be susceptible to further cleavage by plant proteases. Cleavage ofthe active insect toxin at a naturally occurring proteolytic site maylead to proteolytic inactivation of the toxin. As used herein,“proteolytic inactivation” connotes cleavage of the active insect toxinat a naturally occurring proteolytic site by a plant protease, whereincleavage at that site reduces or eliminates the pesticidal activity ofthe insect toxin. In one embodiment, the insect toxin is engineered toreplace a naturally occurring proteolytic site that is sensitive tocleavage by a plant protease with a proteolytic protection site. A“proteolytic protection site” is intended to mean a site that is notsensitive to cleavage by an endogenous plant protease. Replacement of anaturally occurring proteolytic site sensitive to cleavage by a plantprotease with a proteolytic protection site protects the insect toxinfrom proteolytic inactivation by the plant. See co-pending U.S. patentapplication Ser. No. 10/746,914, entitled “Genes Encoding Proteins withPesticidal Activity,” filed Dec. 23, 2003, herein incorporated byreference.

In some embodiments, an insect protoxin is engineered to comprise aproteolytic activation site that is recognized by a novel insect gutprotease. The invention provides nucleic acid molecules, and variantsand fragments thereof, that encode novel insect gut proteases.Specifically, the invention provides nucleic acid molecules encodingnovel proteases identified in the midgut of Diabrotica virgiferavirgifera (i.e., western corn rootworm, hereinafter WCRW). Thenucleotide sequences set forth in SEQ ID NOs:1 and 3 encode novelcysteine proteases that belong to the cathepsin L-like subfamily ofproteases. The nucleotide sequences set forth in SEQ ID NOs:1 and 3encode the polypeptide sequences (i.e., proteases) of SEQ ID NOs:2 and4, respectively. The invention further encompasses variants andfragments of these polypeptide sequences that possess proteolyticactivity as defined herein below. Assays for measuring proteolyticactivity are well known in the art.

Studies indicate that the novel cathepsin L-like proteases of theinvention represent the two most abundant forms of the cathepsin-typeproteases expressed within the WCRW midgut and, therefore, are expectedto be significantly involved in the digestive process. Previous researchhas demonstrated that mammalian cathepsin L-like proteases have ageneral preference for F-R-(A/S/K/N/Q) with cleavage C-terminal to thearginine position. Little is known about the proteolytic cleavagesite(s) for insect pest cathepsin L-like proteases. Thus, the novel WCRWgut proteases of the invention find use, for example, in identifying thepreferred proteolytic cleavage site(s) for these proteases. In anotherembodiment, the insect gut proteases are used to identify proteolyticcleavage sites within pesticidal polypeptides, such as Cry8Bb1 andCry8Bc1, that are susceptible to these proteases.

Knowledge about the preferred proteolytic sites for the insect gutproteases of the invention may lead to improvements in the activationand stability of insect toxins. For example, a proteolytic activationsite that is sensitive to cleavage by an insect gut protease of theinvention may be introduced into an activation region of an insectprotoxin. When this modified protoxin is expressed in a plant and aninsect pest, such as WCRW, feeds on the transgenic plant, the protoxinis cleaved by a cathepsin L-like protease of the invention in the insectgut, thereby producing the active toxin and impacting the insect pest.In one embodiment, the engineered proteolytic activation site issensitive to cleavage by the cathepsin L-like protease of SEQ ID NO:2 or4. In some embodiments, the insect protoxin is Cry8Bb1 or Cry8Bc1.

It is further recognized that insect protoxins or toxins expressed in aplant may be susceptible to cleavage by insect gut proteases uponingestion by an insect pest. Cleavage of an active insect toxin by aninsect gut protease may lead to proteolytic inactivation of the toxin.In this context, “proteolytic inactivation” refers to cleavage of aninsect toxin at a proteolytic site by an insect gut protease, whereincleavage at that site reduces or eliminates the pesticidal activity ofthe toxin. In one embodiment, an insect toxin is engineered to replace aproteolytic site that is sensitive to cleavage by an insect gut proteasewith a proteolytic protection site. By “proteolytic protection site,” asite that is not sensitive to cleavage by an insect gut protease isintended. Replacement of a proteolytic site sensitive to cleavage by aninsect gut protease with a proteolytic protection site protects theinsect toxin from proteolytic inactivation in the insect gut.Eliminating protease-sensitive sites may prevent the insect toxin fromrapid degradation in the insect midgut after ingestion, allowing thetoxin to reach its target intact and more rapidly reach an insecticidaldose within the insect pest. In one embodiment, the proteolyticprotection site is engineered to be insensitive to cleavage by acathepsin L-like protease of the invention, i.e., the polypeptide of SEQID NO:2 or 4. In some embodiments, the insect toxin is Cry8Bb1 orCry8Bc1.

The nucleic acids of the invention encoding the novel cathepsin L-likeinsect gut proteases (SEQ ID NOs:1 and 3) and the polypeptides theyencode (SEQ ID NOs:2 and 4) find further use in identifying anddesigning inhibitors of these proteases. Chemical and biological agentsthat inhibit these proteases could exhibit strong pesticidal effectsupon insect feeding. For example, such inhibitors may result in theinability of the insect pest to digest food and supply the necessarydietary factors needed to support growth and development. In someembodiments, the inhibitors of the novel cathepsin L-like proteases ofthe invention are polypeptides. In a particular embodiment, nucleic acidmolecules encoding the polypeptide inhibitors of the insect gutproteases of the invention are used to generate transgenic plants. Theseplants find use in controlling an insect pest of a plant. In otherembodiments, polypeptide inhibitors of the cathepsin L-like proteases ofthe invention are used to control pests by applying the inhibitorcomposition to the environment of pest.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues (e.g., peptide nucleic acids) having the essential nature ofnatural nucleotides in that they hybridize to single-stranded nucleicacids in a manner similar to naturally occurring nucleotides.

The use of the terms “polynucleotide constructs” or “nucleotideconstructs” herein is not intended to limit the present invention tonucleotide constructs comprising DNA. Those of ordinary skill in the artwill recognize that nucleotide constructs, particularly polynucleotidesand oligonucleotides composed of ribonucleotides and combinations ofribonucleotides and deoxyribonucleotides, may also be employed in themethods disclosed herein. The nucleotide constructs, nucleic acids, andnucleotide sequences of the invention additionally encompass allcomplementary forms of such constructs, molecules, and sequences.Further, the nucleotide constructs, nucleotide molecules, and nucleotidesequences of the present invention encompass all nucleotide constructs,molecules, and sequences which can be employed in the methods of thepresent invention for transforming plants including, but not limited to,those comprised of deoxyribonucleotides, ribonucleotides, andcombinations thereof. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thenucleotide constructs, nucleic acids, and nucleotide sequences of theinvention also encompass all forms of nucleotide constructs including,but not limited to, single-stranded forms, double-stranded forms,hairpins, stem-and-loop structures, and the like.

As used herein, the terms “encoding” or “encoded” when used in thecontext of a specified nucleic acid mean that the nucleic acid comprisesthe requisite information to direct translation of the nucleotidesequence into a specified protein. The information by which a protein isencoded is specified by the use of codons. A nucleic acid encoding aprotein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid or may lack such interveningnon-translated sequences (e.g., as in cDNA).

As used herein, the term “recombinantly engineered” or “engineered” or“modified” connotes the utilization of recombinant DNA technology tointroduce (e.g., engineer) a change in the protein structure based on anunderstanding of the protein's mechanism of action and a considerationof the amino acids being introduced, deleted, or substituted. Forexample, a nucleic acid molecule encoding an insect protoxin may beengineered to comprise a coding sequence for a proteolytic activationsite as described elsewhere herein.

As used herein, “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire nucleicacid sequence or the entire amino acid sequence of a native sequence.“Native sequence” is intended to mean an endogenous sequence, i.e., anon-engineered sequence found in an organism's genome. A full-lengthpolynucleotide encodes the full-length form of the specified protein.

As used herein, the term “antisense” used in the context of orientationof a nucleotide sequence refers to a duplex polynucleotide sequence thatis operably linked to a promoter in an orientation where the antisensestrand is transcribed. The antisense strand is sufficientlycomplementary to an endogenous transcription product such thattranslation of the endogenous transcription product is often inhibited.Thus, where the term “antisense” is used in the context of a particularnucleotide sequence, the term refers to the complementary strand of thereference transcription product.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The terms “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide, or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogues of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

Polypeptides of the invention can be produced either from a nucleic aciddisclosed herein, or by the use of standard molecular biologytechniques. For example, a truncated protein of the invention can beproduced by expression of a recombinant nucleic acid of the invention inan appropriate host cell, or alternatively by a combination of ex vivoprocedures, such as protease digestion and purification.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially or essentially free from components that normallyaccompany or interact with the nucleic acid molecule or protein as foundin its naturally occurring environment. Thus, an isolated or purifiednucleic acid molecule or protein is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

An “isolated” nucleic acid is free of sequences (optimally proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.

As used herein, the term “isolated” or “purified” as it is used to referto a protein of the invention, means that the isolated protein issubstantially free of cellular material, and includes preparations ofprotein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight)of contaminating protein. When the protein of the invention orbiologically active portion thereof is recombinantly produced, optimallyculture medium represents less than about 30%, 20%, 10%, 5%, or 1% (bydry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins (i.e., insect protoxins and insect gut proteases) encodedthereby are also encompassed by the present invention. A “fragment” isintended to mean a portion of a nucleotide sequence of the invention ora portion of an amino acid sequence of a polypeptide of the invention.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein. Hence, fragmentsof an insect protoxin nucleotide sequence may encode protein fragmentsthat become active insect toxins (i.e., possess pesticidal activity)upon cleavage by a protease. In contrast, fragments of an insect gutprotease nucleotide sequence of the invention may encode proteinfragments that have proteolytic activity as described herein andrecognize the preferred proteolytic cleavage site of the nativeprotease. Alternatively, fragments of a nucleotide sequence that areuseful as hybridization probes generally do not encode fragment proteinsretaining biological activity as defined herein above. Thus, fragmentsof a nucleotide sequence may range from at least about 20 nucleotides,about 50 nucleotides, about 100 nucleotides, and up to the full-lengthnucleotide sequence encoding the polypeptides of the invention.

A fragment of a nucleotide sequence of the invention that encodes abiologically active portion of a protein of the invention will encode atleast 15, 25, 30, 50, 100, 200, or 300 contiguous amino acids, or up tothe total number of amino acids present in a full-length polypeptide ofthe invention. Fragments of a nucleotide sequence that are useful ashybridization probes or PCR primers generally need not encode abiologically active portion of a protein of the invention.

Thus, a fragment of a nucleotide sequence of the invention may encode abiologically active portion of a protoxin or insect gut protease, or itmay be a fragment that can be used as a hybridization probe or PCRprimer using methods disclosed below. For example, a biologically activeportion of an insect gut protease can be prepared by isolating a portionof one of the insect gut protease nucleotide sequences of the invention,expressing the encoded portion of the protease (e.g., by recombinantexpression in vitro), and assessing the proteolytic activity of theencoded portion of the insect gut protease

Nucleic acid molecules that are fragments of a nucleotide sequence ofthe invention comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200,1,300, or 1,400 nucleotides, or up to the number of nucleotides presentin a full-length nucleotide sequence disclosed herein

“Variants” is intended to mean substantially similar sequences. Fornucleotide sequences, a variant comprises a deletion and/or addition ofone or more nucleotides at one or more internal sites within the nativenucleotide sequence and/or a substitution of one or more nucleotides atone or more sites in the native nucleotide sequence. As used herein, a“native” nucleotide sequence or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the polypeptides of the invention. Naturallyoccurring allelic variants such as these can be identified with the useof well-known molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivedpolynucleotides, such as those generated, for example, by usingsite-directed mutagenesis but which still encode am insect protoxin orinsect gut protease of the invention. Generally, variants of aparticular nucleotide sequence of the invention will have at least about40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to thatparticular nucleotide sequence as determined by sequence alignmentprograms and parameters described elsewhere herein.

Variants of a particular nucleotide sequence of the invention (i.e., thereference nucleotide sequence) can also be evaluated by comparison ofthe percent sequence identity between the polypeptide encoded by avariant nucleotide sequence and the polypeptide encoded by the referencenucleotide sequence. Thus, for example, isolated nucleic acids thatencode a polypeptide with a given percent sequence identity to an insectprotoxin or insect gut protease of the invention are disclosed. Percentsequence identity between any two polypeptides can be calculated usingsequence alignment programs described elsewhere herein using defaultparameters. Where any given pair of polynucleotides of the invention isevaluated by comparison of the percent sequence identity shared by thetwo polypeptides they encode, the percent sequence identity between thetwo encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore sequence identity.

By “variant” protein, a protein derived from the native protein bydeletion (so-called truncation) or addition of one or more amino acidsto the N-terminal and/or C-terminal end of the native protein; deletionor addition of one or more amino acids at one or more sites in thenative protein; or substitution of one or more amino acids at one ormore sites in the native protein is intended. Variant proteinsencompassed by the present invention are biologically active, that isthey continue to possess the desired biological activity of the nativeprotein. Hence, a variant of an insect protoxin of the invention becomesan active insect toxin (i.e., possesses pesticidal activity) uponcleavage by a protease. In the case of an insect gut protease of theinvention, a variant has proteolytic activity as described herein andrecognizes the preferred proteolytic cleavage site of the nativeprotease. Such variants may result from, for example, geneticpolymorphism or from human manipulation. Biologically active variants ofa native protein of the invention will have at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to the amino acid sequencefor the native protein as determined by sequence alignment programsdescribed elsewhere herein using default parameters. A biologicallyactive variant of a protein of the invention may differ from thatprotein by as few as 1-15 amino acid residues, as few as 1-10, such as6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the proteins of the inventioncan be prepared by mutations in the DNA. Methods for mutagenesis andnucleotide sequence alterations are well known in the art. See, forexample, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel etal. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192;Walker and Gaastra, eds. (1983) Techniques in Molecular Biology(MacMillan Publishing Company, New York) and the references citedtherein. Guidance as to appropriate amino acid substitutions that do notaffect biological activity of the protein of interest may be found inthe model of Dayhoff et al. (1978) Atlas of Protein Sequence andStructure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference. Conservative substitutions, such asexchanging one amino acid with another having similar properties, may bepreferable.

Thus, the nucleotide sequences of the invention include both thenaturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass both naturally occurring proteins aswell as variations and modified forms thereof. Such variants willcontinue to possess the desired biological activity. Obviously, themutations that will be made in the DNA encoding the variant must notplace the sequence out of reading frame and optimally will not createcomplementary regions that could produce secondary mRNA structure. See,EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity of aninsect protoxin of the invention can be evaluated by, for example,insect-feeding assays. See, e.g., Marrone et al. (1985) J. Econ.Entomol. 78:290-293 and Czapla and Lang (1990) supra, hereinincorporated by reference. Assays for assessing the proteolytic activityof an insect gut protease of the invention are well known in the art.

Variant nucleotide sequences also encompass sequences and proteinsderived from a mutagenic and recombinogenic procedure such as DNAshuffling. With such a procedure, one or more different coding sequencescan be manipulated to create a new protein possessing the desiredproperties. In this manner, libraries of recombinant polynucleotides aregenerated from a population of related sequence polynucleotidescomprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. Strategies for suchDNA shuffling are known in the art. See, for example, Stemmer (1994)Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore etal. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl.Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291;and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms, particularly otherinsects. In this manner, methods such as PCR, hybridization, and thelike can be used to identify such sequences based on their sequencehomology to the insect gut protease sequences set forth herein.Sequences isolated based on their sequence identity to an entire insectgut protease sequence set forth herein or to fragments thereof areencompassed by the present invention. Such sequences include sequencesthat are orthologs of the disclosed sequences. By “orthologs,” genesderived from a common ancestral gene and which are found in differentspecies as a result of speciation are intended. Genes found in differentspecies are considered orthologs when their nucleotide sequences and/ortheir encoded protein sequences share substantial identity as definedelsewhere herein. Functions of orthologs are often highly conservedamong species. Thus, isolated sequences that encode an insect gutprotease and which hybridize under stringent conditions to an insect gutprotease sequence disclosed herein, or to fragments thereof, areencompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.),hereinafter “Sambrook.” See also Innis et al., eds. (1990) PCRProtocols: A Guide to Methods and Applications (Academic Press, NewYork); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press,New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual(Academic Press, New York). Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the insect gut proteasesequences of the invention. Methods for preparation of probes forhybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook.

For example, an entire insect gut protease sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding insect gut protease sequencesand messenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique among insectgut protease sequences of the invention and are at least about 10nucleotides in length or at least about 20 nucleotides in length. Suchprobes may be used to amplify corresponding insect gut proteasesequences from a chosen organism, i.e., an insect pest, by PCR. Thistechnique may be used to isolate additional coding sequences from adesired insect pest or as a diagnostic assay to determine the presenceof coding sequences in an insect pest. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook.

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is intended to mean conditions under which a probe willhybridize to its target sequence to a detectably greater degree than toother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length or lessthan 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. for at least 4 hours, more optimally up to 12 hours or longer,and a final wash in 0.1×SSC at 60 to 65° C. for at least about 20minutes. Optionally, wash buffers may comprise about 0.1% to about 1%SDS. Duration of hybridization is generally less than about 24 hours,usually about 4 to about 12 hours. The duration of the wash time will beat least a length sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) (thermal melting point)can be approximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The Tm is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the T_(m) for thespecific sequence and its complement at a defined ionic strength and pH.However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the T_(m); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the T_(m); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe T_(m). Using the equation, hybridization and wash compositions, anddesired T_(m), those of ordinary skill will understand that variationsin the stringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook. Thus, forexample, isolated sequences that encode an insect gut protease of theinvention and which hybridize under stringent conditions to insect gutprotease sequences disclosed herein, or to fragments thereof, areencompassed by the present invention.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity,” and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,U.S.A.). Alignments using these programs can be performed using thedefault parameters. The CLUSTAL program is well described by Higgins etal. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang etal. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol.24:307-331. The ALIGN program is based on the algorithm of Myers andMiller (1988) supra. A PAM120 weight residue table, a gap length penaltyof 12, and a gap penalty of 4 can be used with the ALIGN program whencomparing amino acid sequences. The BLAST programs of Altschul et al(1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin andAltschul (1990) supra. BLAST nucleotide searches can be performed withthe BLASTN program, score=100, wordlength=12, to obtain nucleotidesequences homologous to a nucleotide sequence encoding a protein of theinvention. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al. (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See Altschul et al. (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performedmanually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalentprogram” is intended to mean any sequence comparison program that, forany two sequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad.Sci. USA 89:10915).

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 80%,90%, or 95% sequence identity, compared to a reference sequence usingone of the alignment programs described using standard parameters. Oneof skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, 70%, 80%, 90%, and95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the T_(m) for the specific sequence at a defined ionic strength andpH. However, stringent conditions encompass temperatures in the range ofabout 1° C. to about 20° C., depending upon the desired degree ofstringency as otherwise qualified herein. Nucleic acids that do nothybridize to each other under stringent conditions are stillsubstantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 80%,85%, 90%, or 95% sequence identity to the reference sequence over aspecified comparison window. Optimal alignment is conducted using thehomology alignment algorithm of Needleman and Wunsch (1970) supra. Anindication that two peptide sequences are substantially identical isthat one peptide is immunologically reactive with antibodies raisedagainst the second peptide. Thus, a peptide is substantially identicalto a second peptide, for example, where the two peptides differ only bya conservative substitution. Peptides that are “substantially similar”share sequences as noted above except that residue positions that arenot identical may differ by conservative amino acid changes.

The modified insect protoxin nucleotide sequences of the invention areprovided in expression cassettes for expression in the plant ofinterest. The cassette will include 5′ and 3′ regulatory sequencesoperably linked to a nucleotide sequence of the invention. “Operablylinked” is intended to mean a functional linkage between a promoter anda second sequence, wherein the promoter sequence initiates and mediatestranscription of the DNA sequence corresponding to the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame. The cassette mayadditionally contain at least one additional gene to be cotransformedinto the organism. Alternatively, the additional gene(s) can be providedon multiple expression cassettes.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the protoxin nucleotide sequence to be under thetranscriptional regulation of the regulatory regions. The expressioncassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a modified insect protoxin coding sequence of theinvention, and a transcriptional and translational termination region(i.e., termination region) functional in plants. The promoter may benative or analogous, or foreign or heterologous, to the plant hostand/or to the native insect protoxin nucleotide sequence that isengineered to encode a modified insect protoxin of the invention.Additionally, the promoter may be the natural sequence or alternativelya synthetic sequence. Where the promoter is “foreign” or “heterologous”to the plant host, it is intended that the promoter is not found in thenative plant into which the promoter is introduced. Where the promoteris “foreign” or “heterologous” to the native insect protoxin nucleotidesequence, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked native insectprotoxin nucleotide sequence that has been engineered to encode amodified insect protoxin of the invention. As used herein, a chimericgene comprises a coding sequence operably linked to a transcriptioninitiation region that is heterologous to the coding sequence.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked native protoxinnucleotide sequence that has been engineered, may be native with theplant host, or may be derived from another source (i.e., foreign orheterologous to the promoter, the native protoxin sequence that has beenengineered, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the nucleic acid molecules of the invention may beoptimized for increased expression in the transformed plant. That is, asequence can be synthesized using plant-preferred codons for improvedexpression. See, for example, Campbell and Gowri (1990) Plant Physiol.92:1-11 for a discussion of host-preferred codon usage. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al.(1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picomavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, forexample, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus), and humanimmunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991)Nature 353:90-94); untranslated leader from the coat protein mRNA ofalfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) inMolecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); andmaize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991)Virology 81:382-385). See also, Della-Cioppa et al. (1987) PlantPhysiol. 84:965-968. Other methods known to enhance translation can alsobe utilized, for example, introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones, and2,4-dichlorophenoxyacetate (2,4-D). See generally, Yarranton (1992)Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl.Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff(1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon,pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Sci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724; and U.S. application Ser. Nos.10/004,357; and 10/427,692. Such disclosures are herein incorporated byreference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the present invention.

A number of promoters can be used in the practice of the invention. Thepromoters can be selected based on the desired outcome. That is, thenucleic acids can be combined with constitutive, tissue-preferred, orother promoters for expression in plants. Such constitutive promotersinclude, for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171);ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 andChristensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last etal. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, those disclosedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Generally, it will be beneficial to express the modified insect protoxinsequences from an inducible promoter, particularly from apathogen-inducible promoter. Such promoters include those frompathogenesis-related proteins (PR proteins), which are induced followinginfection by a pathogen; e.g., PR proteins, SAR proteins,beta-1,3-glucanase, chitinase, etc. See, for example, Redolfi et al.(1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also WO99/43819, herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau et al. (1987) PlantMol. Biol. 9:335-342; Matton et al. (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch et al. (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch et al. (1988) Mol. Gen. Genet. 2:93-98; andYang (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen etal. (1996) Plant J. 10:955-966; Zhang et al. (1994) Proc. Natl. Acad.Sci. USA 91:2507-2511; Warner et al. (1993) Plant J. 3:191-201; Siebertzet al. (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible); and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero et al. (1992) Physiol. Mol. Plant Path. 41:189-200).

Additionally, as pathogens find entry into plants through wounds orinsect damage, a wound-inducible promoter may be used in theconstructions of the invention. Such wound-inducible promoters includepotato proteinase inhibitor (pin II) gene (Ryan (1990) Ann. Rev.Phytopath. 28:425-449; Duan et al. (1996) Nature Biotechnology14:494-498); wun1 and wun2, U.S. Pat. No. 5,428,148; win1 and win2(Stanford et al. (1989) Mol. Gen. Genet. 215:200-208); systemin (McGurlet al. (1992) Science 225:1570-1573); WIP1 (Rohmeier et al. (1993) PlantMol. Biol. 22:783-792; Eckelkamp et al. (1993) FEBS Letters 323:73-76);MPI gene (Corderok et al. (1994) Plant J. 6(2):141-150); and the like,herein incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of amodified insect protoxin sequence in a plant through the application ofan exogenous chemical regulator. Depending upon the objective, thepromoter may be a chemical-inducible promoter, where application of thechemical induces gene expression, or a chemical-repressible promoter,where application of the chemical represses gene expression.Chemical-inducible promoters are known in the art and include, but arenot limited to, the maize In2-2 promoter, which is activated bybenzenesulfonamide herbicide safeners, the maize GST promoter, which isactivated by hydrophobic electrophilic compounds that are used aspre-emergent herbicides, and the tobacco PR-1a promoter, which isactivated by salicylic acid. Other chemical-regulated promoters ofinterest include steroid-responsive promoters (see, for example, theglucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl.Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J.14(2):247-257) and tetracycline-inducible and tetracycline-repressiblepromoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet.227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), hereinincorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced modifiedinsect protoxin expression within a particular plant tissue.Tissue-preferred promoters include Yamamoto et al. (1997) Plant J.12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803;Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al.(1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) PlantPhysiol. 112(3):1331-1341; Van Camp et al. (1996) Plant Physiol.112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524;Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994)Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant MolBiol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J.4(3):495-505. Such promoters can be modified, if necessary, for weakexpression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, for example, Hire et al. (1992) Plant Mol.Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene);Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specificcontrol element in the GRP 1.8 gene of French bean); Sanger et al.(1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of themannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao etal. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encodingcytosolic glutamine synthetase (GS), which is expressed in roots androot nodules of soybean). See also Bogusz et al. (1990) Plant Cell2(7):633-641, where two root-specific promoters isolated from hemoglobingenes from the nitrogen-fixing nonlegume Parasponia andersonii and therelated non-nitrogen-fixing nonlegume Trema tomentosa are described. Thepromoters of these genes were linked to a β-glucuronidase reporter geneand introduced into both the nonlegume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes (see Plant Science (Limerick)79(1):69-76). They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teeri et al. (1989)used gene fusion to lacZ to show that the Agrobacterium T-DNA geneencoding octopine synthase is especially active in the epidermis of theroot tip and that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene (see EMBO J. 8(2):343-350). The TR1′ gene, fused tonptII (neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter (Kuster et al. (1995) Plant Mol. Biol. 29(4):759-772); and rolBpromoter (Capana et al. (1994) Plant Mol. Biol. 25(4):681-691. See alsoU.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836;5,110,732; and 5,023,179.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See Thompson et al. (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase); (see WO 00/11177 and U.S. Pat. No.6,225,529, herein incorporated by reference). Gamma-zein is a preferredendosperm-specific promoter. Glob-1 is a preferred embryo-specificpromoter. For dicots, seed-specific promoters include, but are notlimited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin,cruciferin, and the like. For monocots, seed-specific promoters include,but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein,g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO00/12733, where seed-preferred promoters from end1 and end2 genes aredisclosed; herein incorporated by reference. A promoter that has“preferred” expression in a particular tissue is expressed in thattissue to a greater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, a “weak promoter” is intended to mean a promoter that drivesexpression of a coding sequence at a low level. By low level expression,levels of about 1/1000 transcripts to about 1/100,000 transcripts toabout 1/500,000 transcripts is intended. Alternatively, it is recognizedthat weak promoters also encompasses promoters that are expressed inonly a few cells and not in others to give a total low level ofexpression. Where a promoter is expressed at unacceptably high levels,portions of the promoter sequence can be deleted or modified to decreaseexpression levels.

Such weak constitutive promoters include, for example, the core promoterof the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121;5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also,U.S. Pat. No. 6,177,611, herein incorporated by reference.

The methods of the invention involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is intended to meanpresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the invention do not depend on a particular methodfor introducing a sequence into a plant, only that the polynucleotide orpolypeptides gains access to the interior of at least one cell of theplant. Methods for introducing polynucleotide or polypeptides intoplants are known in the art including, but not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” is intended to mean that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al.(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Alsosee Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al.(1987) Particulate Science and Technology 5:27-37 (onion); Christou etal. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl.Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the insect protoxin or insect gut proteasesequences of the invention can be provided to a plant using a variety oftransient transformation methods. Such transient transformation methodsinclude, but are not limited to, the introduction of the insect protoxinor insect gut protease protein or variants and fragments thereofdirectly into the plant or the introduction of the a protein transcriptinto the plant. Such methods include, for example, microinjection orparticle bombardment. See, for example, Crossway et al (1986) Mol Gen.Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler etal. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994)The Journal of Cell Science 107:775-784, all of which are hereinincorporated by reference. Alternatively, the insect protoxin or insectgut protease polynucleotide can be transiently transformed into theplant using techniques known in the art. Such techniques include viralvector system and the precipitation of the polynucleotide in a mannerthat precludes subsequent release of the DNA. Thus, the transcriptionfrom the particle-bound DNA can occur, but the frequency with which itsreleased to become integrated into the genome is greatly reduced. Suchmethods include the use particles coated with polyethylimine (PEI; Sigma#P3143).

In other embodiments, the polynucleotide of the invention may beintroduced into plants by contacting plants with a virus or viralnucleic acids. Generally, such methods involve incorporating anucleotide construct of the invention within a viral DNA or RNAmolecule. It is recognized that the an insect protoxin or insect gutprotease of the invention may be initially synthesized as part of aviral polyprotein, which later may be processed by proteolysis in vivoor in vitro to produce the desired recombinant protein. Further, it isrecognized that promoters of the invention also encompass promotersutilized for transcription by viral RNA polymerases. Methods forintroducing polynucleotides into plants and expressing a protein encodedtherein, involving viral DNA or RNA molecules, are known in the art.See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785,5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

The invention also encompasses transformed or transgenic plantscomprising at least one nucleotide sequence of the invention. Optimally,the plant is stably transformed with a nucleotide construct comprisingat least one nucleotide sequence of the invention operably linked to apromoter that drives expression in a plant cell. As used herein, theterms “transformed plant” and “transgenic plant” refer to a plant thatcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomeof a transgenic or transformed plant such that the polynucleotide ispassed on to successive generations. The heterologous polynucleotide maybe integrated into the genome alone or as part of a recombinantexpression cassette.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part, or plant thegenotype of which has been altered by the presence of heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transposition,or spontaneous mutation.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, andprogeny of same. Parts of transgenic plants are to be understood withinthe scope of the invention to comprise, for example, plant cells,protoplasts, tissues, callus, embryos as well as flowers, ovules, stems,fruits, leaves, roots originating in transgenic plants or their progenypreviously transformed with a DNA molecule of the invention andtherefore consisting at least in part of transgenic cells, are also anobject of the present invention.

As used herein, the term “plant cell” includes, without limitation,seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. The class of plants that can be used in the methods of theinvention is generally as broad as the class of higher plants amenableto transformation techniques, including both monocotyledonous anddicotyledonous plants. Such plants include, for example, Solanumtuberosum and Zea mays.

The present invention may be used for transformation and protection ofany plant species, including, but not limited to, monocots and dicots.Examples of plant species of interest include, but are not limited to,corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava(Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaamericana), fig (Ficus casica), guava (Psidium guajava), mango(Mangifera indica), olive (Olea europaea), papaya (Carica papaya),cashew (Anacardium occidentale), macadamia (Macadamia integrifolia),almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane(Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.), more optimally corn and soybean plants, yet moreoptimally corn plants.

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seedplants include cotton, soybean, safflower, sunflower, Brassica, maize,alfalfa, palm, coconut, etc. Leguminous plants include beans and peas.Beans include guar, locust bean, fenugreek, soybean, garden beans,cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

In the present invention, an isolated modified insect protoxin proteincan be formulated with an acceptable carrier into a protoxin compositionor formulation that is, for example, a suspension, a solution, anemulsion, a dusting powder, a dispersible granule, a wettable powder,and an emulsifiable concentrate, an aerosol, an impregnated granule, anadjuvant, a coatable paste, and also encapsulations in, for example,polymer substances.

Such compositions disclosed above may be obtained by the addition of asurface-active agent, an inert carrier, a preservative, a humectant, afeeding stimulant, an attractant, an encapsulating agent, a binder, anemulsifier, a dye, a UV protectant, a buffer, a flow agent orfertilizers, micronutrient donors, or other preparations that influenceplant growth. One or more agrochemicals including, but not limited to,herbicides, insecticides, fungicides, bactericides, nematicides,molluscicides, acaracides, plant growth regulators, harvest aids, andfertilizers, can be combined with carriers, surfactants or adjuvantscustomarily employed in the art of formulation or other components tofacilitate product handling and application for particular target pests.Suitable carriers and adjuvants can be solid or liquid and correspond tothe substances ordinarily employed in formulation technology, e.g.,natural or regenerated mineral substances, solvents, dispersants,wetting agents, tackifiers, binders, or fertilizers. The activeingredients of the present invention are normally applied in the form ofcompositions and can be applied to the crop area, plant, or seed to betreated. For example, the compositions of the present invention may beapplied to grain in preparation for or during storage in a grain bin orsilo, etc. The compositions of the present invention may be appliedsimultaneously or in succession with other compounds. Methods ofapplying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the modified protoxin proteins of the present invention include,but are not limited to, foliar application, seed coating, and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

Suitable surface-active agents include, but are not limited to, anioniccompounds such as a carboxylate of, for example, a metal; carboxylate ofa long chain fatty acid; an N-acylsarcosinate; mono or di-esters ofphosphoric acid with fatty alcohol ethoxylates or salts of such esters;fatty alcohol sulfates such as sodium dodecyl sulfate, sodium octadecylsulfate or sodium cetyl sulfate; ethoxylated fatty alcohol sulfates;ethoxylated alkylphenol sulfates; lignin sulfonates; petroleumsulfonates; alkyl aryl sulfonates such as alkyl-benzene sulfonates orlower alkylnaphtalene sulfonates, e.g., butyl-naphthalene sulfonate;salts of sulfonated naphthalene-formaldehyde condensates; salts ofsulfonated phenol-formaldehyde condensates; more complex sulfonates suchas the amide sulfonates, e.g., the sulfonated condensation product ofoleic acid and N-methyl taurine; or the dialkyl sulfosuccinates, e.g.,the sodium sulfonate or dioctyl succinate. Non-ionic agents includecondensation products of fatty acid esters, fatty alcohols, fatty acidamides or fatty-alkyl- or alkenyl-substituted phenols with ethyleneoxide, fatty esters of polyhydric alcohol ethers, e.g., sorbitan fattyacid esters, condensation products of such esters with ethylene oxide,e.g., polyoxyethylene sorbitar fatty acid esters, block copolymers ofethylene oxide and propylene oxide, acetylenic glycols such as2,4,7,9-tetraethyl-5-decyn-4,7-diol, or ethoxylated acetylenic glycols.Examples of a cationic surface-active agent include, for instance, analiphatic mono-, di-, or polyamine such as an acetate, naphthenate oroleate; or oxygen-containing amine such as an amine oxide ofpolyoxyethylene alkylamine; an amide-linked amine prepared by thecondensation of a carboxylic acid with a di- or polyamine; or aquaternary ammonium salt.

Examples of inert materials include but are not limited to inorganicminerals such as kaolin, phyllosilicates, carbonates, sulfates,phosphates, or botanical materials such as cork, powdered corncobs,peanut hulls, rice hulls, and walnut shells.

The compositions of the present invention can be in a suitable form fordirect application or as a concentrate of primary composition thatrequires dilution with a suitable quantity of water or other diluantbefore application. The modified insect protoxin concentration will varydepending upon the nature of the particular formulation, specifically,whether it is a concentrate or to be used directly. The compositioncontains 1 to 98% of a solid or liquid inert carrier, and 0 to 50%,optimally 0.1 to 50% of a surfactant. These compositions will beadministered at the labeled rate for the commercial product, optimallyabout 0.01 lb-5.0 lb. per acre when in dry form and at about 0.01pts.-10 pts. per acre when in liquid form.

In a further embodiment, the compositions of the invention can betreated prior to formulation to prolong the pesticidal activity whenapplied to the environment of a target pest as long as the pretreatmentis not deleterious to the activity. Such treatment can be by chemicaland/or physical means as long as the treatment does not deleteriouslyaffect the properties of the composition(s). Examples of chemicalreagents include but are not limited to halogenating agents; aldehydessuch a formaldehyde and glutaraldehyde; anti-infectives, such aszephiran chloride; alcohols, such as isopropanol and ethanol; andhistological fixatives, such as Bouin's fixative and Helly's fixative(see, for example, Humason (1967) Animal Tissue Techniques (W.H. Freemanand Co.).

The protoxin compositions and formulations of the invention can beapplied to the environment of an insect pest by, for example, spraying,atomizing, dusting, scattering, coating or pouring, introducing into oron the soil, introducing into irrigation water, by seed treatment orgeneral application or dusting at the time when the pest has begun toappear or before the appearance of pests as a protective measure. Forexample, the modified insect protoxin protein of the invention may bemixed with grain to protect the grain during storage. It is generallyimportant to obtain good control of pests in the early stages of plantgrowth, as this is the time when the plant can be most severely damaged.The compositions of the invention can conveniently contain anotherinsecticide if this is thought necessary. In an embodiment of theinvention, the composition is applied directly to the soil, at a time ofplanting, in granular form of a composition of a carrier. Anotherembodiment is a granular form of a composition comprising anagrochemical such as, for example, a herbicide, an insecticide, afertilizer, or an inert carrier.

Compositions of the invention find use in protecting plants, seeds, andplant products in a variety of ways. For example, the compositions canbe used in a method that involves placing an effective amount of themodified insect protoxin composition in the environment of the pest by aprocedure selected from the group consisting of spraying, dusting,broadcasting, or seed coating.

Before plant propagation material (fruit, tuber, bulb, corm, grains,seed), but especially seed, is sold as a commercial product, it iscustomarily treated with a protectant coating comprising herbicides,insecticides, fungicides, bactericides, nematicides, molluscicides, ormixtures of several of these preparations, if desired together withfurther carriers, surfactants, or application-promoting adjuvantscustomarily employed in the art of formulation to provide protectionagainst damage caused by bacterial, fungal, or animal pests. In order totreat the seed, the protectant coating may be applied to the seedseither by impregnating the tubers or grains with a liquid formulation orby coating them with a combined wet or dry formulation. In addition, inspecial cases, other methods of application to plants are possible,e.g., treatment directed at the buds or the fruit.

The plant seed of the invention comprising a DNA molecule comprising anucleotide sequence encoding a modified protoxin protein of theinvention may be treated with a seed protectant coating comprising aseed treatment compound, such as, for example, captan, carboxin, thiram,methalaxyl, pirimiphos-methyl, and others that are commonly used in seedtreatment. In one embodiment within the scope of the invention, a seedprotectant coating comprising a pesticidal composition of the inventionis used alone or in combination with one of the seed protectant coatingscustomarily used in seed treatment.

The embodiments of the present invention may be effective against avariety of pests. For purposes of the present invention, pests include,but are not limited to, insects, fungi, bacteria, nematodes, acarids,protozoan pathogens, animal-parasitic liver flukes, and the like. Pestsof particular interest are insect pests, particularly insect pests thatcause significant damage to agricultural plants. “Insect pests” isintended to mean insects and other similar pests such as, for example,those of the order Acari including, but not limited to, mites and ticks.Insect pests of the present invention include, but are not limited to,insects of the order Lepidoptera, e.g. Achoroia grisella, Aclerisgloverana, Acleris variana, Adoxophyes orana, Agrotis ipsilon, Alabamaargillacea, Alsophila pometaria, Amyelois transitella, Anagastakuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi,Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara,Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneurasp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydialatiferreanus, Cydia pomonella, Datana integerrima, Dendrolimussibericus, Desmia feneralis, Diaphania hyalinata, Diaphania nitidalis,Diatraea grandiosella, Diatraea saccharalis, Ennomos subsignaria,Eoreuma loftini, Esphestia elutella, Erannis tilaria, Estigmene acrea,Eulia salubricola, Eupocoellia ambiguella, Eupoecilia ambiguella,Euproctis chrysorrhoea, Euxoa messoria, Galleria mellonella, Grapholitamolesta, Harrisina americana, Helicoverpa subflexa, Helicoverpa zea,Heliothis virescens, Hemileuca oliviae, Homoeosoma electellum, Hyphantiacunea, Keiferia lycopersicella, Lambdinafiscellaria fiscellaria,Lambdina fiscellaria lugubrosa, Leucoma salicis, Lobesia botrana,Loxostege sticticalis, Lymantria dispar, Macalla thyrisalis, Malacosomasp., Mamestra brassicae, Mamestra configurata, Manduca quinquemaculata,Manduca sexta, Maruca testulalis, Melanchra picta, Operophtera brumata,Orgyia sp., Ostrinia nubilalis, Paleacrita vernata, Papilio cresphontes,Pectinophora gossypiella, Phryganidia californica, Phyllonorycterblancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendana, Platynota stultana, Platyptilia carduidactyla, Plodiainterpunctella, Plutella xylostella, Pontia protodice, Pseudaletiaunipuncta, Pseudoplasia includens, Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera sp.,Thaurnstopoea pityocampa, Tinsola bisselliella, Trichoplusia hi, Udearubigalis, Xylomyges curiails, and Yponomeuta padella.

Also, the embodiments of the invention may be effective against avariety of insect pests including insects selected from the ordersColeoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera,Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura,Siphonaptera, Trichoptera, etc., particularly Coleoptera andLepidoptera. Insect pests of the invention for the major crops include:Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, blackcutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fallarmyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; chinch bug, e.g., Blissus leucopterus leucopterus; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Jylemyaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Vrevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, crucifer flea beetle; Phyllotretastriolata, striped flea beetle; Phyllotreta nemorum, striped turnip fleabeetle; Meligethes aeneus, rapeseed beetle; and the pollen beetlesMeligethes rufimanus, Meligethes nigrescens, Meligethes canadianus, andMeligethes viridescens; Potato: Leptinotarsa decemlineata, Coloradopotato beetle.

Furthermore, embodiments of the present invention may be effectiveagainst Hemiptera such as Lygus hesperus, Lygus lineolaris, Lyguspratensis, Lygus rugulipennis Popp, Lygus pabulinus, Calocorisnorvegicus, Orthops compestris, Plesiocoris rugicollis, Cyrtopeltismodestus, Cyrtopeltis notatus, Spanagonicus albofasciatus, Diaphnocorischlorinonis, Labopidicola allii, Pseudatomoscelis seriatus, Adelphocorisrapidus, Poecilocapsus lineatus, Blissus leucopterus, Nysius ericae,Nysiusraphanus, Euschistus servus, Nezara viridula, Eurygaster,Coreidae, Pyrrhocoridae, Tinidae, Blostomatidae, Reduviidae, andCimicidae. Pests of interest also include Araecerus fasciculatus, coffeebean weevil; Acanthoscelides obtectus, bean weevil; Bruchus rufimanus,broadbean weevil; Bruchus pisorum, pea weevil; Zabrotes subfasciatus,Mexican bean weevil; Diabrotica balteata, banded cucumber beetle;Cerotoma trifurcata, bean leaf beetle; Diabrotica virgifera, Mexicancorn rootworm; Epitrix cucumeris, potato flea beetle; Chaetocnemaconfinis, sweet potato flea beetle; Hypera postica, alfalfa weevil;Anthonomus quadrigibbus, apple curculio; Sternechus paludatus, beanstalk weevil; Hypera brunnipennis, Egyptian alfalfa weevil; Sitophilusgranaries, granary weevil; Craponius inaequalis, grape curculio;Sitophilus zeamais, maize weevil; Conotrachelus nenuphar, plum curculio;Euscepes postfaciatus, West Indian sweet potato weevil; Maladeracastanea, Asiatic garden beetle; Rhizotrogus majalis, European chafer;Macrodactylus subspinosus, rose chafer; Tribolium confusum, confusedflour beetle; Tenebrio obscurus, dark mealworm; Tribolium castaneum, redflour beetle; Tenebrio molitor, yellow mealworm.

Nematodes include plant-parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera and Globodera spp. such asGlobodera rostochiensis and Globodera pailida (potato cyst nematodes);Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beetcyst nematode); and Heterodera avenae (cereal cyst nematode).

Units, prefixes, and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The above-defined terms are more fullydefined by reference to the specification as a whole.

The following examples are presented by way of illustration, not by wayof limitation.

EXPERIMENTAL Example 1 Transformation of Maize and Regeneration ofTransgenic Plants

The coding sequence for a full-length Cry8Bb1 protoxin (SEQ ID NO:5) ismodified to comprise codons for a proteolytic activation site.Specifically, a DNA sequence encoding the FRRGFRRG (SEQ ID NO:6)proteolytic peptide is introduced in the junction between the N-terminalcrystalline forming segment of the Cry8Bb1 protoxin and the C-terminalportion of the protoxin that comprises the active insect toxin uponcleavage. Immature maize embryos from greenhouse donor plants arebombarded with a plasmid containing the modified Cry8Bb1 protoxinnucleotide sequence operably linked to ubiquitin promoter and theselectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37),which confers resistance to the herbicide Bialaphos. Alternatively, theselectable marker gene is provided on a separate plasmid. Transformationis performed as follows. Media recipes follow below.

Preparation of Target Tissue

The ears are husked and surface sterilized in 30% Clorox bleach plus0.5% Micro detergent for 20 minutes, and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

Preparation of DNA

A plasmid vector comprising the modified Cry8Bb1 protoxin nucleotidesequence described above, operably linked to a ubiquitin promoter, ismade. This plasmid DNA plus plasmid DNA containing a PAT selectablemarker is precipitated onto 1.1 μm (average diameter) tungsten pelletsusing a CaCl₂ precipitation procedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

100 μl 2.5 M CaCl₂

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for expression of the modified Cry8Bb1protoxin by assays known in the art, such as, for example, immunoassaysand western blotting.

Analysis of Transgenic Maize Plants

Transgenic maize plants positive for expression of the modified Cry8Bb1protoxin are tested for resistance to WCRW using standard bioassaysknown in the art. Such methods include, for example, root excisionbioassays and whole plant bioassays. See, e.g., U.S. Patent PublicationNo. US 2003/0120054 and International Publication No. WO 03/018810.

Bombardment and Culture Media Recipes

560Y Bombardment Medium

-   4.0 g/L N6 basal salts (SIGMA C-1416)-   1.0 mL/L Eriksson's Vitamin Mix (1000X SIGMA-1511)-   0.5 mg/L thiamine HCl-   120.0 g/L sucrose-   1.0 mg/L 2,4-D-   2.88 g/L L-proline

Ingredients are mixed and brought to volume with D-I H₂O followingadjustment to pH 5.8 with KOH. Gelrite is then added to a concentrationof 2.0 g/L Gelrite and the medium is sterilized and cooled to roomtemperature. Finally, 8.5 mg/L silver nitrate is added.

560R Selection Medium

-   4.0 g/L N6 basal salts (SIGMA C-1416)-   1.0 mL/L Eriksson's Vitamin Mix (1000X SIGMA-1511)-   0.5 mg/L thiamine HCl-   30.0 g/L sucrose-   2.0 mg/L 2,4-D    Ingredients are mixed and brought to volume with D-I H₂O following    adjustment to pH 5.8 with KOH. Gelrite is then added to a    concentration of 3.0 g/L and the medium is sterilized and cooled to    room temperature. Finally, 0.85 mg/L silver nitrate and 3.0 mg/L    bialaphos are added.    288J Plant Regeneration Medium-   4.3 g/L MS salts (GIBCO 11117-074)-   5.0 mL/L MS vitamins stock solution (0.100 g nicotinic acid, 0.02    g/L thiamine HCL, 0.10 g/L pyridoxine HCl, and 0.40 g/L glycine    brought to volume with polished D-I H₂O) (Murashige and Skoog (1962)    Physiol. Plant. 15:473)-   100 mg/L myo-inositol-   0.5 mg/L zeatin-   60 g/L sucrose-   1.0 mL/L 0.1 mM abscisic acid    Ingredients are mixed and brought to volume with D-I H₂O following    adjustment to pH 5.6 with KOH. Gelrite is then added to a    concentration of 3.0 g/L and the medium is sterilized and cooled to    60° C. Finally, 1.0 mg/L indoleacetic acid and 3.0 mg/L bialaphos    are added.    Hormone-Free Medium (272V)-   4.3 g/L MS salts (GIBCO 11117-074)-   5.0 mL/L MS vitamins stock solution (supra)-   0.1 g/L myo-inositol-   40.0 g/L sucrose    Ingredients are mixed and brought to volume with D-I H₂O following    adjustment to pH 5.6 with KOH. Bacto-agar is then added to a    concentration of 6.0 g/L and the medium is sterilized and cooled to    60° C.

Example 2 Agrobacterium-Mediated Transformation of Maize andRegeneration of Transgenic Plants

For Agrobacterium-mediated transformation of maize with the modifiedCry8Bb1 protoxin nucleotide sequence of Example 1, the method of Zhao isemployed (U.S. Pat. No. 5,981,840, and PCT patent publicationWO98/32326; the contents of which are hereby incorporated by reference).Briefly, immature embryos are isolated from maize and the embryoscontacted with a suspension of Agrobacterium, where the bacteria arecapable of transferring the modified Cry8Bb1 protoxin nucleotidesequence to at least one cell of at least one of the immature embryos(step 1: the infection step). In this step the immature embryos areimmersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). Optimally the immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Optimally the immature embryos arecultured on solid medium with antibiotic, but without a selecting agent,for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Optimally, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium are cultured on solid medium to regenerate the plants.Transgenic maize plants positive for expression of the modified Cry8Bb1protoxin are tested for resistance to WCRW, as described in Example 1.

Example 3 Proteolytic Cleavage of a Modified Insect Toxin in TransgenicPlants

The portion of the Cry8Bb1 loop between helix 3 and helix 4 of domain 1of the insecticidal toxin, which has the sequence NGSR (SEQ ID NO: 7),was replaced by a protease cathepsin-L motif that has a sequence ofFRRGFRRG (SEQ ID NO: 6). A construct containing this modified sequencewas used to transform maize plants. Maize plants were transformed usingthe Agrobacterium protocol outlined in Example 2.

After transformation, transgenic maize plants expressing the modifiedCry8Bb1 toxin protein were analyzed for the stability of the toxinmolecule. Western analysis of the transgenic leaf and root tissuesindicated that the toxin molecule generated in planta was cleaved bymaize proteases into three fragments in the leaf and root tissues.

In order to characterize the cleavage sites, immunoprecipitation of thefragments using AminoLink (Pierce, Rockford Ill.) was initiated. Theresulting fragments from the immunoprecipitation were separated bySDS-page electrophoresis and blotted into PVD membrane. Three proteinbands of interest were cut and sequenced. The highest band fragment,which was also the least intense, indicated that the toxin molecule wasintact. The sequence of the next fragment indicated that the toxinmolecule was proteolytically cleaved in the plant, resulting in theremoval of the first 49 amino acids. The lowest band sequence, which wasalso the most prevalent, showed that the toxin molecule was cleavedwithin the protease cathepsin-L motif FRRGFRRG (SEQ ID NO:6) at the lastR.

The cleavage by maize root and leaf proteases of the modified Cry8Bb1toxin molecule was primarily at this site. It was therefore concludedthat maize proteases have high affinity for the protease motif FRRGFRRG(SEQ ID NO:6). Classical Cry proteins have a toxin domain and acrystal-forming domain. Only the toxin domain is needed for insecticidalactivity. This motif was then introduced into the boundary of the toxindomain of Cry8Bb1 and its crystal-forming domain in order to obtainprocessing by plant proteases. The resulting cleavage at FRRGFRRG (SEQID NO:6) by plant proteases is expected to result in two proteinproducts, the active toxin domain and the non-active crystal domain.

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

All publications, patents and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications, patents and patentapplications are herein incorporated by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the embodiments described herein.

1. A method for protecting a plant from an insect pest, said methodcomprising introducing into said plant at least one polynucleotideconstruct that comprises a nucleotide sequence encoding Bacillusthuringiensis Cry8Bb1 protoxin operably linked to a promoter that drivesexpression in said plant, wherein said Cry8Bb1 protoxin has at least oneproteolytic activation site that has been engineered to comprise acleavage site that is sensitive to a plant protease that is presentwithin said plant, wherein said proteolytic activation site comprisesthe amino acid sequence set forth in SEQ ID NO:6, wherein expression ofsaid polynucleotide construct produces said Cry8Bb1 protoxin in saidplant, wherein cleavage of said Cry8Bb1 protoxin by said plant proteaseproduces an active Cry8Bb1 toxin, and wherein said active Cry8Bb1 toxinprotects said plant from said insect pest.
 2. The method of claim 1,wherein said active Cry8Bb1 toxin comprises at least one proteolyticprotection site that has been engineered to comprise a site that is notsensitive to cleavage by said plant protease, wherein said proteolyticprotection site replaces a naturally occurring proteolytic site that issensitive to said plant protease, and wherein said proteolyticprotection site protects said active Cry8Bb1 toxin from proteolyticinactivation by said plant protease.
 3. The method of claim 1, whereinsaid insect pest is selected from the group consisting of Coloradopotato beetle, western corn rootworm, southern corn rootworm, and bollweevil.
 4. The method of claim 1, wherein said plant is a monocot. 5.The method of claim 4, wherein said monocot is maize.
 6. The method ofclaim 1, wherein said plant is a dicot.
 7. The method of claim 1,wherein said plant protease is a cysteine protease.
 8. The method ofclaim 7, wherein said cysteine protease is a cathepsin B-like protease.9. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding a Bacillus thuringiensis Cry8Bb1 protoxin, wherein said Cry8Bb1protoxin has at least one proteolytic activation site that has beenengineered to comprise a cleavage site that is sensitive to a plantprotease that is present within a plant, wherein said proteolyticactivation site comprises the amino acid sequence set forth in SEQ IDNO:6, and wherein cleavage of said Cry8Bb1 protoxin by said plantprotease produces an active Cry8Bb1 toxin.
 10. An expression cassettecomprising the nucleic acid molecule of claim 9 operably linked to apromoter that drives expression in a plant.
 11. A transformed plantcomprising the expression cassette of claim
 10. 12. The plant of claim11, wherein said expression cassette is stably incorporated into thegenome of the plant.
 13. A transformed seed of the plant of claim 12,wherein the seed comprises the expression cassette.